ANALYTICAL BIOCHEMISTRY 241, 151–155 (1996)ARTICLE NO. 0392

Preparation of Neoglycolipids with a Definite Sugar ChainMoiety from Glycoproteins1

Yasuko Ozaki, Kaoru Omichi,2 and Sumihiro HaseDepartment of Chemistry, Osaka University College of Science, Machikaneyama-cho 1-1, Toyonaka, Osaka 560, Japan

Received March 26, 1996

tures, and chromatographic purification of sugarA method for preparing neoglycolipids possessing a chains before conjugation or of neoglycolipids prepared

sugar chain with a definite structure using two N- is, in practice, difficult. An alternative route for prepar-linked sugar chains as model compounds was devel- ing neoglycolipids with a definite sugar chain is to useoped. Pyridylaminated sugar chains obtained from pyridylaminated (PA-)3 sugar chains, which can be eas-glycoproteins were purified by reversed-phase HPLC ily purified and separated from one another by re-and a pyridylaminated sugar chain thus obtained was versed-phase HPLC.converted to a 1-amino-1-deoxy derivative. The prod- This paper describes the latter method of neoglyco-uct was conjugated by reductive amination with lipid preparation, using two PA-sugar chains expressedphosphatidylglycolaldehyde prepared by periodate mainly in mouse brain (3) as model compounds.oxidation of dipalmitoyl phosphatidylglycerol. Neo-glycolipids formed were purified by gel filtration from

MATERIALS AND METHODSthe reaction mixture. The structures of the purifiedneoglycolipids were confirmed by composition analy- Materialssis for sugar, fatty acid, phosphorus, and 2-acetamido-

Dipalmitoyl phosphatidylglycerol was purchased1,2-dideoxy-1-[(hydroxyethyl)amino]-D-glucitol, the link-from Sigma (St. Louis, MO). Hen egg yolk antibody,age region of the sugar chain, and lipid moieties. TheIgY, was prepared from hen egg yolk as reported (4).method provides a convenient means of preparing neo-b-Galactosidase (Aspergillus oryzae) and b-N-acetyl-glycolipids having a definite structure. q 1996 Academic

Press, Inc. hexosaminidase (Diplococcus pneumoniae) were pur-chased from Toyobo (Osaka, Japan) and BoehringerMannheim (Mannheim, Germany), respectively. Cos-mosil columns 5C18-P (4.6 1 150 mm) and 5SL (4.6 1The roles of the sugar chain moiety in glycolipids are 150 mm) were obtained from Nacalai Tesque (Kyoto,studied by using the lipid moiety as an immobilized Japan). Toyopearl HW-40F was a product of Tosohprobe on silica gel or plastic plates, or as a microparti- (Tokyo, Japan). Thin-layer plates (DC-Alufolien Kiesel-culate probe after incorporation into liposomes. Neogly- gel 60 and DC-Plastikfolien Kieselgel 60) were pur-colipids prepared with sugar chains from glycoproteins chased from Merck (Darmstadt, Germany). Tri-Sil wasare readily amenable to assay procedures established purchased from Pierce (Rockford, IL) and Lens culi-for glycosphingolipids. The conjugation of oligosaccha- naris agglutinin conjugated with fluorescein isothiocy-rides released from glycoproteins with phosphatidyl- anate (FITC-LCA) from Seikagaku Kogyo (Tokyo, Ja-ethanolamine has been reported (1, 2). However,

because sugar moieties of glycoproteins are usuallyheterogeneous, the neoglycolipids thus prepared are a 3 Abbreviations used: PA-, pyridylamino; FITC, fluorescein isothio-

cyanate; LCA, Lens culinaris agglutinin; BA-1, GlcNAcb1–2Mana1–6mixture of moieties with various sugar chain struc-(GlcNAcb1–4)(Mana1–3)Manb1–4GlcNAcb1–4(Fuca1–6)GlcNAc;BA-2, GlcNAcb1–2Mana1–6(GlcNAcb1–4)(GlcNAcb1–2Mana1–3)-Manb1–4GlcNAcb1–4(Fuca1–6)GlcNAc; GlcNAc, N-acetyl-D-glu-1 This study was supported in part by a Grant-in-Aid for Scientific

Research on Priority Areas from the Ministry of Education, Science, cosamine; FAB–MS, fast atom bombardment–mass spectrometry;PG, phosphatidylglycerol; Hepes, N-2-hydroxyethylpiperazine-N *-2-Sports and Culture of Japan.

2 To whom correspondence should be addressed. Fax: /81 6 ethanesulfonic acid; GLC, gas–liquid chromatography; Tris, tris(hy-droxymethyl)aminomethane; Fuc, L-fucose; Man, D-mannose.8505383.

1510003-2697/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

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OZAKI, OMICHI, AND HASE152

pan). Authentic BA-1-PA and BA-2-PA were prepared acid. Palladium black (4 mg) was added to the solution,and then hydrogen gas was blown into the solutionas reported (3). 2-Acetamido-1,2-dideoxy-1-[(2-hy-

droxyethyl)amino]-D-glucitol was prepared by reduc- with gentle stirring for 3 h at room temperature. Thefluorescence disappeared upon reduction of the PA-tive amination of GlcNAc with ethanolamine (at 607C,

for 16 h) and purified by gel filtration on a Toyopearl sugar chain. After removal of palladium black by cen-trifugation, the supernatant was lyophilized. To thisHW-40F column (1.8 1 121 cm) equilibrated with 10

mM ammonium acetate buffer, pH 6.0. The elution was 0.2 ml of anhydrous hydrazine was added, and the solu-tion was heated at 707C for 2 min. Excess hydrazinemonitored by TLC. Its molecular weight determined by

FAB–MS was 267.1 (calculated for C10H22O6N2, 267.1). was removed by repeated evaporation with toluene invacuo. The residue was dissolved in 1 ml of 10 mM

ammoniun acetate buffer, pH 6.0. 1-Amino-1-deoxy de-Preparation of BA-1-PA and BA-2-PA from IgYrivatives (BA-1-NH2, BA-2-NH2) were purified by gelSugar chains were liberated from IgY (100 mg) by filtration on a Toyopearl HW-40F column (1.3 1 105hydrazinolysis (1007C, 10 h) followed by N-acetylation. cm) equilibrated with 10 mM ammonium acetate buffer,The reducing ends of the sugar chains liberated were pH 6.0. The elution was monitored by ninhydrin re-pyridylaminated as reported previously (5). The pH of agent and methanol–sulfuric acid on TLC plates.the reaction mixture was brought to 10 with 6 M ammo-

nium hydroxide solution, and then excess 2-aminopyri-Preparation and Purification of Neoglycolipidsdine was extracted five times with an equal volume of

chloroform. After pH adjustment to 6.0 with acetic acid, Dipalmitoyl phosphatidylglycerol sodium salt (PG,500 nmol) in 50 ml of a mixture of methanol and 0.2 Mthe water phase was concentrated to a small volume,

and chromatographed on a Toyopearl HW-40F column acetic acid solution (1:1, v/v) was mixed with 20 ml ofa 0.1 M sodium metaperiodate solution in 0.2 M sodium(5.5 1 35 cm) equilibrated with 10 mM ammonium ace-

tate buffer, pH 6.0, to remove residual reagents. The acetate buffer, pH 5.0, and the mixture was then keptat 07C for 20 min in the dark. Ten microliters of thePA-sugar chain fraction was hydrolyzed with 2 ml of

0.1 M hydrochloric acid at 807C for 45 min to remove reaction mixture was applied onto a HPLC column(Cosmosil 5SL, 4.6 1 150 mm) to isolate the oxidizedsialic acid residues. The pH of the solution was ad-

justed to neutral with 6 M ammonium hydroxide solu- PG (phosphatidylglycolaldehyde). Elution was done us-ing a mixture of methanol, acetonitrile and watertion and the solution was then lyophilized. The residue

was dissolved in 2 ml of 50 mM ammonium acetate (7:4:3, v/v) as an eluent at a flow rate of 1.0 ml/min.The elution of oxidized PG was monitored by the ab-buffer, pH 5.0, and digested with b-galactosidase (350

units) at 377C for 20 h. BA-2-PA was purified from the sorbance at 207 nm.BA-1-NH2 or BA-2-NH2 (30 nmol) in 10 ml of 0.1 Mdigest by repetition of HPLC on a Cosmosil 5C18-P

column (4.6 1 150 mm). Solvents A and B were used. Hepes buffer, pH 7.5, and 10 ml of 0.5 M sodium cyano-borohydride in methanol were added to 333 ml of theSolvent A comprised 0.1 M ammonium acetate buffer,

pH 4.0, and solvent B comprised 0.1 M ammonium ace- oxidized PG solution prepared from 10 ml of the reac-tion mixture. The mixture was concentrated to abouttate buffer, pH 4.0, containing 0.5% 1-butanol. The col-

umn was equilibrated with 5% solvent B. After injec- 40 ml under reduced pressure and the solution was thenallowed to stand for 16 h at 257C. The reaction mixturetion of a sample, the proportion of solvent B was pro-

grammed to increase from 5 to 100% in 55 min at a was applied onto a Toyopearl HW-40F column (0.8 140 cm) equilibrated with 50 mM ammonium acetateflow rate of 1.5 ml/min. Elution was monitored by the

fluorescence at 400 nm (excitation wavelength, 320 nm). buffer, pH 6.0, and the elution was monitored by TLCand GLC.BA-1-PA was prepared by digestion of 200 nmol of

BA-2-PA with 0.1 units of b-N-acetylhexosaminidase(D. pneumoniae) in 2.8 ml of 50 mM ammonium acetate TLC Analysis of Neoglycolipidsbuffer, pH 5.0, at 377C for 5 h as reported previously The neoglycolipids isolated by gel filtration were ana-(3). BA-1-PA was purified by HPLC as described above. lyzed by TLC using chloroform, methanol, and water

(50:55:18, v/v/v) as a solvent. TLC plates developedConversion of BA-2-PA and BA-1-PA to 1-Amino-1- were treated with the orcinol reagent or FITC–LCA

deoxy Derivatives conjugate. Detection with the FITC–LCA conjugatewas carried out as follows: A developed TLC plate (DC-Conversion of BA-2-PA and BA-1-PA to 1-amino-1-

deoxy derivatives was carried out according to the plastikfolien) was soaked in the FITC–LCA conjugatesolution (200 mg in 1 ml of 10 mM Tris–HCl buffer, pHmethod reported previously (6) as follows: Each PA-

sugar chain (500 nmol) was dissolved in 1 ml of water, 8.0, containing 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2,and 0.15 M NaCl) and incubated at 257C for 30 min.and the pH of the solution was adjusted to 4 with acetic

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NEOGLYCOLIPIDS WITH A DEFINITE SUGAR CHAIN MOIETY 153

FIG. 2. Purification of periodate oxidized PG by HPLC. PG wasoxidized with periodate and the reaction mixture was chromato-

FIG. 1. Purification of BA-2-PA by HPLC. The b-galactosidase di- graphed on a Cosmosil 5SL column. The fraction indicated by a bargest of asialo sugar chains from IgY was separated by reversed-phase was collected as oxidized PG. The arrowhead indicates the elutionHPLC. The arrowhead indicates the elution position of authentic position of PG. The peak eluted between 1 and 2 min was due toBA-2-PA. The peak indicated by a bar was collected. excess reagents.

as BA-2-PA by reversed-phase and size-fractionationAfter the plate was washed three times with the sameHPLC using authentic sugar chains (3) and by sugarTris–HCl buffer, the FITC–LCA conjugate bound tocomposition analysis (data not shown) (8). The yield ofneoglycolipids on the plate was detected under a uvBA-2-PA was 500 nmol from 100 mg of IgY. BA-2-PAlamp at 365 nm. The elution position of lipids was de-was converted to BA-2-NH2, which was purified by geltected by Dittmer–Lester reagent (7).filtration. BA-1-PA was prepared by digestion of BA-2-

Composition Analyses of Neoglycolipids PA with b-N-acetylhexosaminidase and BA-1-NH2 wasprepared from BA-1-PA in the same way as describedNeoglycolipids were hydrolyzed with 4 M trifluoro-for BA-2-NH2. The yields of 1-amino-1-deoxy sugaracetic acid at 1007C for 3 h. Monosaccharides were ana-chains from PA-sugar chains were almost quantitative.lyzed by HPLC as PA-sugars by the method previously

reported (8). Neutral sugars, palmitate, and 2-acet- Preparation of Neoglycolipidsamido - 1,2 - dideoxy - 1 - [(2 - hydroxyethyl)amino] - D - PG was oxidized with sodium metaperiodate and theglucitol were assayed by GLC on a 2% OV-17 column oxidized PG was purified by HPLC (Fig. 2). The fraction(0.3 cm1 2 m) after methanolysis (1 M methanolic HCl,at 907C for 18 h) followed by trimethylsilylation withTri-Sil (9). Phosphorus was assayed by the method ofBartlett (10).

RESULTS AND DISCUSSION

Preparation of BA-1-NH2 and BA-2-NH2 from IgYThe model compounds we selected were BA-1 and

BA-2, which are mainly expressed in mouse brain.However, because the amounts obtained from mousebrain are insufficient for the preparation of neoglyco-conjugates, BA-1-NH2 and BA-2-NH2 were preparedfrom egg yolk IgY, which contains a sialylfucosylbian-tennary sugar chain with a bisecting GlcNAc residueas a major sugar chain (11) and is easily isolated fromhen egg yolk. Liberation of the sugar chains from IgY FIG. 3. Purification of BA-2-PG by gel filtration. The reaction mixture

of BA-2-NH2 and oxidized PG was applied to a Toyopearl HW-40Fand pyridylamination were performed as described un-column (0.8 1 40 cm) equilibrated with 50 mM ammonium acetateder Materials and Methods. After mild acid hydrolysisbuffer, pH 6.0, and eluted with the same buffer. A part of each fractionfollowed by b-galactosidase digestion of PA-sugar was methanolyzed followed by GLC analysis. Arrows indicate the elu-

chains, BA-2-PA was purified by reversed-phase HPLC tion position: a, BA-2-NH2; and b, NaBH3CN. A similar result wasobtained for the purification of a neoglycolipid from BA-1-NH2.(Fig. 1). The peak indicated by a bar was identified

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OZAKI, OMICHI, AND HASE154

TABLE 1

Component Analysis of Neoglycolipids

Component BA-2-PG BA-1-PG

GlcNAc 3.9 3.1Man 3.0a 3.0a

Fuc 1.1 1.3P 0.8 0.9Palmitate 2.0 1.92-Acetamido-1,2-dideoxy-

1-[(2-hydroxyethyl)amino]-D-glucitol 1.0 0.9

Note. GlcNAc was quantified by HPLC after acid hydrolysis. Phos-phorus was measured by the method of Bartlett. Neutral sugars,palmitate, and 2-acetamido-1,2-dideoxy-1-[(2-hydroxyethyl)amino]-D-glucitol were assayed by GLC after methanolysis followed by tri-methylsilylation.

a The value of mannose was taken as 3.0.

Structural Confirmation of Neoglycolipids

The neoglycolipids prepared, BA-2-PG and BA-1-PG,were analyzed by TLC (Fig. 5). The fluorescent spotsdetected with the FITC–LCA conjugate had the samemobilities as those detected with the orcinol reagent,

FIG. 4. Scheme for neoglycolipid preparation. R1|(CH2)14CH3{, indicating the presence of a lipid and the sugar chain

R2|GlcNAcb1–2Mana1–6(GlcNAcb1–4)(GlcNAcb1–2Mana1–3)-

on the same spot. Each sample gave one spot and theManb1–4GlcNAcb1– or GlcNAcb1–2Mana1–6(GlcNAcb1–4)(Mana1–3)-positions to which they migrated were between the un-Manb1–4GlcNAcb1–.reacted lipid and sugar chains. A composition analysisof the neoglycolipids is given in Table 1. The values arein good agreement with the expected structures.indicated by a bar contained both phosphorus and pal-Furthermore, 2-acetamido-1,2-dideoxy-1-[(hydroxyethyl)-mitate, and it was collected as oxidized PG, which wasamino]-D-glucitol, the linkage region of sugar chain andthen coupled with BA-2-NH2 or BA-1-NH2. The reac-lipid moieties, was detected by GLC analysis (Fig. 6).tion mixture was separated by gel filtration (Fig. 3).From these results, the neoglycolipids prepared wereThe fractions indicated by a bar contained both man-considered to be the structures shown in Fig. 4. Thenose and palmitate, and were collected as neoglyco-total yield of neoglycolipids from 1-amino-1-deoxy de-lipid. BA-2-NH2 or BA-1-NH2 was not detected, whilerivatives was about 85%.residual oxidized PG was eluted at fraction numbers

30–33. The reaction scheme for the conjugation isshown in Fig. 4.

FIG. 5. TLC of prepared neoglycolipids. (a) Lane 1, BA-2-NH2; (a) FIG. 6. GLC of the methanolysate of BA-2-PG. BA-2-PG was meth-anolyzed and trimethylsilylated prior to GLC. The arrowheads indi-lane 2 and (b) lane 3, BA-2-PG; (c) lane 1, BA-1-NH2; (c) lane 2 and

(d) lane 3, BA-1-PG. a and c were stained with the orcinol reagent, cate the elution positions of standard compounds: a, Fuc; b, Man; c,glucitol (internal standard); d, palmitate; e, 2-acetamido-1,2-dideoxy-and b and d were visualized with the FITC–LCA conjugate under a

uv-lamp. The arrowheads indicate the migration positions of oxidized 1-[(2-hydroxyethyl)amino]-D-glucitol (detected as 2-amino-1,2-di-deoxy-1-[(2-hydroxyethyl)amino]-D-glucitol).PG detected by Dittmer–Lester reagent.

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NEOGLYCOLIPIDS WITH A DEFINITE SUGAR CHAIN MOIETY 155

2. Feizi, T., Stoll, M. S., Yuen, C.-T., Chai, W., and Lawson, A. M.Neoglycolipids possessing the same structure can be(1994) Methods Enzymol. 230, 484–519.prepared by conjugation of BA-1 and BA-2 with phos-

3. Shimizu, H., Ochiai, K., Ikenaka, K., Mikoshiba, K., and Hase,phatidylethanolamine by reductive amination (1, 2),S. (1993) J. Biochem. 114, 334–338.but the sugar chains released from IgY were a mixture

of various structures and the resulting neoglycolipids 4. Hatta, H., Kim, M., and Yamamoto, T. (1990) Agric. Biol. Chem.54, 2531–2535.were heterogeneous with respect to their sugar struc-

tures, for which no effective purification step is readily 5. Kuraya, N., and Hase, S. (1992) J. Biochem. 112, 122–126.available. The method described here is a convenient

6. Hase, S. (1992) J. Biochem. 112, 266–268.route for preparing neoglycolipids having a definite7. Dittmer, J. C., and Lester, R. L. (1964) J. Lipid Res. 5, 126–sugar structure, taking advantage of the fact that PA-

127.sugar chains are easily purified by reversed-phase8. Hase, S., Hatanaka, K., Ochiai, K., and Shimizu, H. (1992) Bi-HPLC and that conversion of a PA-sugar chain into the

osci. Biotech. Biochem. 56, 1676–1677.amino derivative is possible. The neoglycolipids BA-2-PG and BA-1-PG can be used as antigens for BA-2 and 9. Sweeley, C. C., Bentley, R., Makita, M., and Wells, W. W. (1963)BA-1, respectively, because they have homogeneous J. Am. Chem. Soc. 85, 2497–2507.sugar chains and lipid moieties. 10. Bartlett, G. R. (1959) J. Biol. Chem. 234, 466–468.

11. Ohta, M., Hamako, J., Yamamoto, S., Hatta, H., Kim, M., Yama-REFERENCES moto, T., Oka, S., Mizuochi, T., and Matsuura, F. (1991) Glyco-

conjugate J. 8, 400–413.1. Stoll, M. S., Mizuochi, T., Childs, R. A., and Feizi, T. (1988)Biochem. J. 256, 661–664.

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